We investigate the molecular mechanisms by which transmembrane proteins (referred to as cargo) are sorted to different compartments of the endomembrane system in eukaryotic cells. This system comprises an array of membrane-enclosed organelles including the endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network (TGN), endosomes, lysosomes, lysosome-related organelles (LROs) (e.g., melanosomes), and different domains of the plasma membrane in polarized cells (e.g., epithelial cells and neurons). Transport of cargo between these compartments is mediated by carrier vesicles or tubules that bud from a donor compartment, translocate through the cytoplasm, and eventually fuse with an acceptor compartment. Work in our laboratory focuses on the molecular machineries that mediate these processes, including (1) sorting signals and adaptor proteins that select cargo proteins for packaging into the transport carriers, (2) microtubule motors and organelle adaptors that drive movement of the transport carriers and other organelles through the cytoplasm, and (3) tethering factors that promote fusion of the transport carriers to acceptor compartments. These machineries are studied in the context of different intracellular transport pathways, including endocytosis, recycling to the plasma membrane, retrograde transport from endosomes to the TGN, biogenesis of lysosomes and LROs, and polarized sorting in epithelial cells and neurons. Knowledge gained from this research is applied to the elucidation of disease mechanisms, including congenital disorders of protein traffic such as the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS) and the neurocutaneous disorder MEDNIK syndrome, neurodegenerative disorders such as Alzheimers disease, and the exploitation of intracellular transport by pathogens such as HIV-1. Function in the BORC complex in the regulation of lysosome movement - The multiple functions of lysosomes are critically dependent on their ability to move bidirectionally along microtubules between the center and the periphery of the cell. Centrifugal and centripetal movement of lysosomes is mediated by kinesin and dynein motors, respectively. We recently discovered a multi-subunit complex named BORC that recruits the small GTPase Arl8 to lysosomes to promote their kinesin-dependent movement toward the cell periphery. We showed that BORC and Arl8 function upstream of two structurally distinct kinesin types: kinesin-1 (KIF5B) and kinesin-3 (KIF1B and KIF1A). Remarkably, KIF5B and KIF1B/KIF1A move lysosomes along different microtubule tracks. These findings thus established BORC as a master regulator of lysosome positioning through coupling to different kinesins and microtubule tracks. The BORC complex coordinates encounter and fusion of lysosomes with autophagosomes - Whereas the mechanisms involved in autophagosome formation have been extensively studied for the past two decades, those responsible for autophagosome-lysosome fusion have only recently begun to garner attention. We recently found that BORC is also required for efficient autophagosome-lysosome fusion. Knock out (KO) of BORC subunits impairs both the encounter and fusion of autophagosomes with lysosomes. Reduced encounters result from an inability of lysosomes to move toward the peripheral cytoplasm, where many autophagosomes are formed. BORC KO also reduces the recruitment of the HOPS tethering complex to lysosomes and assembly of a trans-SNARE complex involved in autophagosome-lysosome fusion. Through these dual roles, BORC integrates the kinesin-dependent movement of lysosomes toward autophagosomes with HOPS-dependent autophagosome-lysosome fusion. Polarized organelle segregation in neurons by differential interactions with microtubule motors - Polarized sorting of newly synthesized proteins to the somatodendritic and axonal domains of neurons occurs by selective incorporation into distinct populations of vesicular transport carriers. We recently found that segregation of these carriers to their corresponding neuronal compartments occurs at a region in the axon hillock named the pre-axonal exclusion zone (PAEZ) though differential coupling to different microtubule motors. We also discovered a chain of interactors including Rab5, the FHF complex and dynein-dynactin that retrieves somatodendritic proteins from the axon, thus contributing to their somatodendritic distribution at steady state. Finally, we demonstrated that an ensemble composed of BORC, Arl8, SKIP, and kinesin-1 specifically drives lysosome transport into the axon, and not the dendrites, in cultured rat hippocampal neurons. We additionally found that this transport is essential for maintenance of axonal growth-cone dynamics and autophagosome turnover. These findings illustrated how a general mechanism for lysosome dispersal in non-neuronal cells is adapted to drive polarized transport in neurons, and emphasized the importance of this mechanism for critical axonal processes. A role for AP-1 in sorting presenilin-2 to late endosomes and lysosomes - In collaboration with Wim Annaert (VIB Center for the Biology of Disease, Leuven, Belgium), we demonstrated a role for the AP-1 complex in the sorting of a novel form of gamma-secretase to late endosomes and lysosomes. Gamma-secretases are a family of intramembrane-cleaving proteases involved in the pathogenesis of Alzheimer's disease. We identified a sorting motif in the cytosolic tail of the presenilin-2 (PSEN2) subunit of gamma-secretase that targets this enzyme to late endosomes and lysosomes. This motif is recognized in a phosphorylation-dependent manner by AP-1. PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates a prominent pool of intracellular amyloid-beta peptide. These findings reveal potentially important roles for lysosome-generated amyloid-beta peptide in Alzheimers disease. Mechanism of cargo recognition by the retromer complex Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer's disease and Parkinson's disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes had remained elusive. X-ray crystallographic, biochemical and cellular studies done in collaboration with Aitor Hierro (CIC bioGUNE, Bilbao, Spain) showed that cargos bind to a cooperative assembly of the VPS26 and VPS35 subunits of retromer in complex with the sorting nexin SNX3. Identification of TSSC1 as a novel component of the endosomal retrieval machinery - Endosomes function as a hub for multiple protein sorting events, including retrograde transport to the trans-Golgi network (TGN) and recycling to the plasma membrane. These processes are mediated by tubular-vesicular carriers that bud from early endosomes and fuse with a corresponding acceptor compartment. We previously investigated the role of two multisubunit tethering complexes named GARP and EARP that participate in SNARE-dependent fusion of endosome-derived carriers with the TGN and recycling endosomes, respectively. We have now discovered that a previously uncharacterized WD40/beta-propeller protein named TSSC1 is a specific interactor of both GARP and EARP, and a novel component of the endosomal retrieval machinery. Interference with TSSC1 impairs both retrograde transport to the TGN and retrieval to the plasma membrane. These findings contribute to the understanding of the pathogenesis of progressive cerebello-cerebral atrophy type 2, a neurodegenerative disorder caused by mutations in the shared Vps53 subunit of GARP and EARP.